Replication of Co1E1 DNA. Studies on DNA replication of plasmid Co1E1 and its relatives have been continued. Transcripts by RNA polymerase (RNA II) that start the 555 nucleotides upstream of the replication origin of plasmid Co1E1 extend beyond the origin. About half of the transcripts form a hybrid with the template DNA. The hybridized transcript is cleaved by ribonuclease H at the origin and used as the primer for DNA synthesis by DNA polymerase I. The primer formation is dependent on the secondary structure of RNA II. Each single base-change affects initiation of synthesis of RNA II, hybrid formation between RNA II and the template DNA, the cleavage sites of RNA II by RNase H, the stability of association of RNA II or the use of cleaved RNA II as a primer by DNA polymerase I. Primer formation is regulated by a plasmid-specified small RNA (RNA I). RNA I binds to RNA II at the complementary region. This binding results in inhibition of formation of the secondary structure necessary for primer formation. Kinetic analysis of binding of RNA I to RNA II revealed that the binding starts at the 5'-end of RNA I and propagates stepwise to its 3'-end. Primer formation is also regulated by a 63-amino acid protein specified by the plasmid. The protein, called Rom, stimulates binding of RNA I to RNA II and thus enhances the inhibitory action of RNA I. Inhibition of primer formation by RNA I in the presence or absence of the protein determines the copy number of a plasmid in a cell and the incompatibility between related plasmids. The region of RNA II that is complementary to RNA I is not required for the primer formation but necessary for its regulation.